Cloning And Expression Of Porcine Reproductive And Respiration Syndrome Virus And The Initial Establishment Of ELISA

Highly pathogenic porcine reproductive and respiratory syndrome which caused by porcine reproductive and respiratory syndrome virus mutant is an acute infectious disease of pigs that sustained high fever as the main characteristics,and its distinguishing feature is the high morbidity and high mortality rates.Nsp2 gene is the largest genetic variation among the highly pathogenic porcine reproductive and respiratory syndrome and the common's isolates.In this study,porcine reproductive and respiratory syndrome virus (PRRSV) mutant SCM were used to clone amplified a section of the Nsp2 gene,then analysising the sequence of the obtained purpose gene,construction of the prokaryotic expression vectors,prokaryotic expression and the initial establishment of the ELISA detection methods.Ⅰ,Cloning and sequence analysis of Nsp2 geneAccording to the reference the Nsp2 gene nucleotide sequence of PRRSV mutant SCJX01,we designed and synthesized a pair of specific primers by Oligo6.0,and extracted the total RNA of PRRSV mutant which can be cultured in Marc-145 cells as a template, amplified the purpose fragments by RT-PCR,recovered the product of PCR,then successfully transfer to E.Coli DH5a.The gene fragment was cloned in the pMD18-T vector by TA cloning method to construct recombinant clones Plasmid pMD-Nsp2,The positive recombinant Plasmid was sented to the company for identifying sequence after the correct digestion by PCR and restriction enzyme.sequencing revealed that the length of the cloned gene fragment in Nsp2 is 523bp,and encoding 166 amino acids.Carrying out bio-informatics Analysis using The sequencing results and Amino acid sequence with the different isolates at home and abroad.Sequence analysis shows that the nucleotide homology is 64.7%by the obtained Nsp2 gene compared with the standard strain VR2332 and vaccine strains MLV.With the mutant compared to the separation of nucleotides sequence homology at 89.4%～94.6%.With other isolated mutants compared,the nucleoside homology at 99.4%～99.8%.As proved that the Nsp2 gene is easily variable, and up till now the manifest about missing gene is almostly same in existing mutants. However,it's relatively conservative compared with a high-deletion region of Nsp2.Ⅱ,Prokaryotic Expression of Nsp2 geneAccording to the obtained recombinant plasmid pMD-Nsp2 and the prokaryotic expression vector pET-32a(+),adding two restriction sites of EcoRⅠand SacⅠ,then both of them which digested by the two enzymes will be connected for constructing the prokaryotic expression plasmid pET-32a(+)-Nsp2.The positive plasmid should be chosen and transformed into BL21(DE3),then be induced to express by 0.5mmol/L IPTG according the best laboratory conditions have been carried out.The expression products is verifed by sodium dodecyl sulfate polyacrylamide gel electrophoresis aroylamino (SDS-PAGE) and immunoblotting experiments(Western blot) that the correct size of the product is and the immuno-reactive of the protein,while detecting it's soluble.The results show that the prokaryotic expression plasmid pET-32a(+)-Nsp2 existed in form of the inclusion body and is about the size of 38kD,the percent is22.5%in total bacterial protein. The expressed products is hightest at 37℃,5h.Ⅲ,Nsp2 gene's protein purification and the initial establishment of ELISA detectionThe recombinant protein which expressed the prokaryotic expression products pET-32a(+)-Nsp2 of the porcine reproductive and respiratory syndrome virus(PRRSV) mutant Nsp2 and exists in form of inclusion body is Purified.Being the pET-32a(+)-Nsp2 recombinant protein as a package Antigen after purification,Optimizing conditions in antigen-coated,reaction time and choice of substrate,Finally we test the effect of this ELISA through the specificity experiment,the duplicated experiment,blocks examination and contrasting with the standard reagent kit.We establish initially the detection method of porcine reproductive and respiratory syndrome virus(PRRSV) mutant by indirect ELISA.The research results shows that optimum concentration of the recombinant antigen coated is 17.5ug/mL,the most conditions suitable for coating is 4℃overnight,serum dilution is 1:40,the seized serum and the reaction time of antibody is respectively 30min and 40min at 37℃,TMB solution as substrate colored 37℃10 min.After statistics analysis, the definite masculine and feminine elements's marginal value is 0.33.And through some testing experiments show that the ELISA has detective specificity and good duplication, high sensitivity;the ELISA method that we established is compared with American IDEXX Co.PRRSV antibody kit,the result is its relative specificity and the sensitivity respectively are 98.1%and 91.5%,the coincidence rate achieves 90.0%,the test result non-significance difference.On this experiment,we cloned and expressed a gene fragment of the PRRSV mutant's Nsp2,and compared with other strains's Nsp2 at home and abroad.while the initial establishment of the ELISA detection method.From some aspects as the bio-information,molecular biology,immunogenicity and so on,we researched into Nsp2 gene,that providing the basic data for more detailed study on gene function of Nsp2 and the variation principle of PRRSV.